Our research is focused on elucidating the mechanisms that underlie ATP-dependent protein remodeling carried out by molecular chaperone machines and the role of chaperones in ATP-dependent proteolysis. Molecular chaperones function during non-stress conditions to facilitate folding of newly synthesized proteins, to remodel protein complexes, and to target regulatory proteins and misfolded proteins for degradation. During cell stress, chaperones play an essential role in preventing folding intermediates from becoming irreversibly damaged and forming protein aggregates. They promote recovery from stress by disaggregating and reactivating proteins. They are also involved in delivering damaged proteins to compartmentalized proteases. Protein aggregation, misfolding and premature degradation are major contributors to a large number of human diseases, including cancer. The goal of our research is to understand how chaperones function and to provide the foundation for discovering preventions and treatments for diseases involving protein misfolding. One aim is to understand the mechanism of action of Hsp90. The Hsp90 family of heat shock proteins represents one of the most abundantly expressed and highly conserved families of molecular chaperones. Eukaryotic Hsp90 is known to control the stability and the activity of more than 200 client proteins, including receptors, protein kinases and transcription factors. Hsp90 is also important for the growth and survival of cancer cells and drugs targeting Hsp90 are currently in clinical trials. To gain insight into the mechanism of action of this important family of chaperones, we are studying Hsp90 from Escherichia coli, Hsp90Ec, and from yeast, Hsp82. We have shown that Hsp90Ec interacts directly with the E. coli Hsp70, DnaK, and functions with DnaK and the DnaK cochaperones to reactivation aggregated proteins. Using a bacterial two-hybrid assay to screen for Hsp90Ec mutants that fail to interact with DnaK, we identified several mutants with amino acid substitutions in the middle domain of Hsp90Ec. The mutant Hsp90Ec proteins were isolated found to be defective in interaction with DnaK and were defective in protein reactivation with the DnaK system. A homologous S. cerevisiae Hsp82 mutant was constructed and found to support cell growth. However, the mutant protein was defective in protein reactivation in vitro, suggesting the same region of Hsp90 is important for chaperone function in E. coli and in yeast. A second aim is to elucidate the mechanism of protein disaggregation by Clp/Hsp100 molecular chaperones, including ClpB of prokaryotes and its yeast homolog, Hsp104. Understanding how energy-dependent protein disaggregating machines function is clinically relevant since protein aggregation is linked to medical conditions, including Alzheimer's disease, Parkinson's disease, amyloidosis and prion diseases. ClpB/Hsp104 promotes cell survival following stress by disaggregating protein aggregates and reactivating stress-inactivated proteins. ClpB and Hsp104 act with a second molecular chaperone system, DnaK in E. coli and Hsp70 in yeast. This teamwork is specific: E. coli DnaK interacts with the E. coli ClpB and yeast Hsp70, Ssa1, interacts with the yeast Hsp104. We showed the interaction is between the M-domains of hexameric ClpB/Hsp104 and the DnaK/Hsp70 nucleotide-binding domain (NBD). To identify the site on E. coli DnaK that interacts with ClpB, we substituted amino acid residues throughout the DnaK NBD. We found that several variants with substitutions in subdomain IB and IIB of the DnaK NBD were defective in interactions with ClpB both in vivo and in vitro. We observed that GrpE, which also interacts with subdomains IB and IIB, inhibited the interaction between ClpB and DnaK in vitro, suggesting competition between ClpB and GrpE for binding DnaK. Additionally, homologous substitutions in subdomains IB and IIB of Ssa1 caused defects in collaboration between Ssa1 and Hsp104. Altogether, these results provide insight into the molecular mechanism of collaboration between the DnaK/Hsp70 system and ClpB/Hsp104 for protein disaggregation. In yeast the Hsp104 chaperone functions with the Hsp70 system to promote prion propagation and to resolubilize and reactivate stress-inactivated proteins. It was previously shown that ClpB and DnaK of E. coli can cooperate with a yeast Hsp40, Sis1, to propagate prions and with another yeast Hsp40, Ydj1, to support yeast thermotolerance. In collaboration with the Masison Laboratory (NIDDK) we identified structural elements that determine the distinct functions of Sis1 and Ydj1 by assessing the function of Sis1-Ydj1 hybrid proteins in various cellular processes. We found that the C-terminal regions of Sis1 and Ydj1 mediated functional distinctions that directed the action of the Hsp104 disaggregation machinery in different processes both in vivo and in reactivation reactions in vitro. A third aim is to investigate the mechanism of action of Clp chaperones in proteolysis. Clp proteases of prokaryotes have analogous structures, functions and mechanisms of action to the eukaryotic proteasome. They are composed of an ATP-dependent protein unfolding component and a protease component. ClpXP is a two-component ATP-dependent protease that unfolds and degrades proteins bearing specific recognition signals. One substrate we showed to be degraded by Escherichia coli ClpXP is FtsZ, an essential cell division protein. FtsZ forms polymers that assemble into a large ring-like structure, termed the Z-ring, during cell division at the site of constriction. To better understand substrate selection by ClpXP, we engineered FtsZ mutant proteins containing amino acid substitutions or deletions near the FtsZ C-terminus. We identified two discrete regions of FtsZ important for degradation of both FtsZ monomers and polymers by ClpXP in vitro. One region is located 30 residues away from the C-terminus and the other region is near the FtsZ C-terminus, partially overlaping the recognition sites for several other FtsZ-interacting proteins, including MinC, ZipA and FtsA. Mutation of either region caused the protein to be more stable and mutation of both caused an additive effect, suggesting that ClpX recognizes FtsZ through dual contacts and residues in both regions are important for degradation by ClpXP. We also observed that in vitro MinC inhibits degradation of FtsZ by ClpXP, suggesting that some of the same residues in the C-terminal site that are important for degradation by ClpXP are important for binding MinC. Some Clp protease are regulated by adaptor proteins and antiadaptor proteins. In an ongoing collaboration with Susan Gottesman's laboratory (NCI), we are studying how ClpXP is regulated by adaptor and anti-adaptor proteins. RssB is a ClpXP adaptor protein that specifically targets RpoS, the stationary phase sigma factor of E. coli, for degradation during exponential growth. In response to stress, RpoS degradation ceases. Three small anti-adaptor proteins, IraP, IraM and IraD, are each made under a different stress condition and interact with RssB to block RpoS degradation. We have been studying the reaction in vitro with purified proteins. By characterizing wild type and mutant RssB proteins we are gaining insight into the mechanism of action of the RssB adaptor and how interaction with antiadaptor proteins inhibits the action of RssB.